PO.BCS01.15 · 生物信息与计算

慢性淋巴细胞白血病患者CD8 T细胞的经济型全长转录组揭示BTKi治疗对mRNA加工和基因表达的影响

Cost-effective, full-length transcriptome of chronic lymphocytic leukemia patient cd8 t cells reveals the impact of BTKi treatment on mRNA processing and gene expression

编号 1509 展板 16 时间 4/20 09:00–12:00 区域 Section 6 主讲 Pearlly Yan, PhD

分会场 Sequence Analysis

查看 PDF 下载 PDF 🔒 查看 / 下载完整 PDF 需登录并开通下载套餐 · 查看套餐 / 开通 AACR 官方页面

作者与单位 Authors & Affiliations

Shanmugapriya Thangavadivel¹, Altan Turkoglu¹, Lianbo Yu¹, Rosario Distefano¹, Alexander Pan², Logan Walker³, Tina Wang⁴, Shrilekha Misra¹, Tzung-Huei Lai¹, Britten Gordon¹, Samon Benrashid¹, Alexander He¹, Meixiao Long¹, rosa Lapalombella¹, John C. Byrd⁵, Kerry A. Rogers¹, Bradley Blaser¹, Ralf Bundschuh⁶, Jennifer A. Woyach¹, Pearlly S. Yan¹

¹The Ohio State University College of Medicine, Columbus, OH,²N-Power Medicine, Inc, Red Wood City, CA,³University of Michigan, Ann Arbor, MI,⁴The Ohio State University, Columbus, OH,⁵University of Pittsburgh Medical Center, Pittsburgh, PA,⁶Department of Physics and Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH

摘要 Abstract

中文摘要

伊布替尼（Ibrutinib）是一种布鲁顿酪氨酸激酶抑制剂（BTKi），用于治疗慢性淋巴细胞白血病（CLL）。它不仅影响CLL细胞，还影响免疫微环境，尤其是T细胞。虽然T细胞耗竭和激活缺陷已被充分研究，但mRNA加工对转录组和免疫组的作用仍需探究。我们和其他研究者注意到CLL细胞中存在此类事件，即较短的异构体取代较长的初级转录本。它们通过改变mRNA稳定性、翻译效率和蛋白质定位实现额外的调控。为节省宝贵的患者时间序列样本，我们开发了一种方案，将300个分选的活细胞置入含有5'模板转换引物和3' poly-dT引物的冷裂解缓冲液中，从而绕过RNA提取并捕获全长转录本。在基因组浏览器中查看数据时，我们注意到比对读段轨道和连接读段轨道以基因特异性方式在患者内部及患者之间揭示了外显子丢失事件，表现为一段段未被覆盖的外显子。为进行异构体知情的基因表达分析，我们为来自10例CLL患者（30个样本：基线、伊布替尼治疗3年和5年）的每个基因确定一个涵盖所有转录外显子的已知异构体，用于长度标准化的基因表达分析。在本摘要中，我们进行了全局分析以及针对来自免疫通路和CD8 T细胞激活的187个基因的子集分析。对于时间点分析，对无外显子丢失的基因和有外显子丢失的基因分别进行差异表达基因（DEG）分析。187基因的时间点分析则在一个列表中进行。主要发现为：i）子集分析得出的DEG也见于两项全局分析中；ii）三项分析中各时间点之间观察到相似的DEG趋势：BL > 3y 和 BL > 5y 最高，其次为 BL > 3y 和 5y > 3y，另有少量 3y > BL 和 5y > BL 事件；iii）88%的表达转录本含有一个或多个外显子丢失事件；iv）使用子集分析DEG的STRING分析显示，CD8激活基因（如ptprc和cd69）与Perforin和PD-1通路基因广泛相互作用，形成一个致密网络，而来自FAS、mTOR和TRAIL通路的基因形成一个相互作用较少的独立网络。总之，在成本与大体RNA-seq相近的情况下，我们的全长数据揭示了长基因（如jak1，26个外显子，30个样本中有12个实现全外显子覆盖）和短基因（如jun，该无内含子基因的一部分在某些样本中未被覆盖）中的mRNA加工事件。300个治疗前后细胞的超低量输入使得对无外显子丢失的高表达基因和有多处丢失的低表达基因均能进行深度文库采样。最后，子集分析得出的DEG支持了这一异构体知情工作流程在全局DEG分析中捕获CD8 T细胞功能基因的能力。

查看英文原文 English abstract

Ibrutinib, a Bruton's tyrosine kinase inhibitor (BTKi), is used to treat chronic lymphocytic leukemia (CLL). It has effects not only on CLL cells but also on the immune microenvironment, particularly T cells. While T cell exhaustion and activation defects are well studied, the role of mRNA processing on the transcriptome and immunome needs investigation. We and others noted such events in CLL cells with shorter isoforms replacing the longer primary transcripts. They enable added regulations via altered mRNA stability, translational efficiency, and protein localization.To conserve precious patient time series samples, we developed a protocol that deposes 300 sorted live cells into cold lysis buffer containing 5' template-switching primers and 3' poly-dT primers to bypass RNA extraction and to capture full-length transcripts. Viewing our data in genome browser, we noted the aligned- and the junction-read tracks reveal exon dropout events as stretches of uncovered exons in a gene-specific manner within- and across patients. For isoform-informed gene expression analysis, we identify a known isoform for each gene that encompasses all the transcribed exons from 10 CLL patients (30 samples: baseline, 3y and 5y ibrutinib treatment) for length-normalized gene expression analysis. For this abstract, we performed global analysis and a subset analysis targeting 187 genes from immune pathways and CD8 T cell activation. For timepoint analysis, differentially expressed gene (DEG) analysis was performed separately for genes without exon dropout and those with exon dropout. The 187-gene timepoint analysis was performed in one list. Key findings are: i) DEGs from subset analysis are also found in the two global analyses; ii) similar DEG trends observed across timepoints in all 3 analyses: highest in BL > 3y and BL > 5y followed by BL > 3y and 5y > 3y with a few 3y > BL and 5y > BL events; iii) 88% of the expressed transcripts contain one or more exon dropout event; iv) STRING analysis using DEGs from subset analysis reveals CD8 activation genes such as ptprc and cd69 interact extensively with Perforin- and PD-1 pathway genes forming a dense network while genes from FAS-, mTOR- and TRAIL pathways form a separate network with few interactions. In summary, with a cost similar to bulk RNA-seq, our full-length data reveals mRNA processing events in both long genes (e.g., jak1 , 26 exons, full exon coverage in 12/30 samples) and short genes (e.g., jun , part of the intronless gene not covered in some samples). An ultralow input of 300 pre- and post-treatment cells allow deep library sampling of both highly expressed genes with no exon dropout and lowly expressed genes with many dropouts. Lastly, DEGs from subset analysis lend support to the ability of the isoform-informed workflow to capture CD8 T cell functional genes in global DEG analysis.

利益披露 Disclosure

S. Thangavadivel, None.. A. Turkoglu, None.. L. Yu, None.. R. Distefano, None.. A. Pan, None.. L. Walker, None.. T. Wang, None.. S. Misra, None.. T. Lai, None.. B. Gordon, None.. S. Benrashid, None.. A. He, None.. M. Long, None.. R. Lapalombella, None. J. C. Byrd, Vincerx Pharma Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly traded company in the past 24 months. Eilean Therapeutics Other, Consultancy (Includes expert testimony). Kurome Therapeutics Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly traded company in the past 24 months. Newave Pharmaceutical Inc. Patent, Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly traded company in the past 24 months. Abbvie Patent, Other, Consultancy (Includes expert testimony). AstraZeneca Patent, Other, Consultancy (Includes expert testimony). Syndax Patent, Other, Consultancy (Includes expert testimony). K. A. Rogers, Alpine Immune Science Patent, Other, Consultancy (Includes expert testimony). AstraZeneca Pharmaceuticals LP Other, Consultancy (Includes expert testimony). Janssen Biotech Inc Patent, Other, Consultancy (Includes expert testimony). Pharmacyclics LLC, an Abbie Company Patent, Other, Consultancy (Includes expert testimony). AbbVie Company Patent, Other, Consultancy (Includes expert testimony). BeiGene Ltd Patent, Consultancy (Includes expert testimony). Genentech, a member of the Roche Group Patent, Consultancy (Includes expert testimony). Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company Patent, Consultancy (Includes expert testimony). Novartis Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly-traded company in the past 24 months. B. Blaser, None.. R. Bundschuh, None. J. A. Woyach, Schrodinger Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly-traded company in the past 24 months. Janssen Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly-traded company in the past 24 months. Pharmacyclics Patent, Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly-traded company in the past 24 months. Newave Patent, Other, Consultancy (Includes expert testimony). Astrazenaca Patent, Other, Consultancy (Includes expert testimony). Merck Patent, Other, Consultancy (Includes expert testimony). BeiGene Patent, Other, Consultancy (Includes expert testimony). Loxo Lilly Patent, Other, Consultancy (Includes expert testimony). Genentech, Inc. Patent, Other, Consultancy (Includes expert testimony). Abbvie Other, Consultancy (Includes expert testimony). Morphosys Other, Consultancy (Includes expert testimony); Divested equity in a private or publicly-traded company in the past 24 months. P. S. Yan, None.

← 返回 AACR 2026 检索